The broad, long-term objectives of this program are to determine (1) the mechanism by which cadmium (Cd) causes bone loss, (2) the basis for increased sensitivity of ovariectomized females to Cd-induced bone loss, and (3) the relevance of our findings to human exposures to Cd. Experiments are designed to test the hypothesis that Cd can act directly on bone or on the hematopoietic precursors to the cells that break down bone, and that these effects are separate from any effects of Cd on other organs (e.g., kidneys or gastrointestinal tract). The first specific aim is to complete a study of long-term Cd in elderly, female beagles. In this study, Cd increased bone resorption within 96 h at 2 to 15 mug/L in blood, well within reported ranges for humans exposed to Cd in cigarettes or industrial settings. An important remaining step is to test whether responses to Cd in bone marrow cultures in vitro (see below) occurred in vivo. Additional analyses will determine whether Cd influenced bone indirectly by causing (1) decreases in the gastrointestinal absorption of calcium, (2) renal tubular dysfunction, (3) decreases in circulating copper concentrations, and (4) pathological changes in adrenal or pituitary glands. The second specific aim is to study the mechanism by which Cd increases the formation/differentiation of multinucleated osteoclast-like (MN-OS) cells from mononuclear bone marrow cells in culture. In this system, significant effects occur at 10 nM Cd (1 ppb), the same concentration as that found in the plasma of the beagles exposed to Cd in vivo. Proposed studies will test whether (1) Cd causes MN-OS cells to acquire biochemical properties and bone-resorbing capabilities of true osteoclasts, (2) Cd acts by stimulating the release of osteotropic cytokines and/or prostaglandin E2, (3) the effector molecule in the bone marrow culture system is Cd bound to metallothionein, and (4) bone marrow cells from ovariectomized animals are more sensitive to Cd. A third specific aim is to conduct a fetal rat limb bone study that parallels study (2) in the marrow culture system, investigating the role of cytokines in the dramatic bone resorption response to 10 nM Cd. The proposed research is critical to current NIH interests in womens' health issues. Results indicate that pregnant, nursing, and postmenopausal women have an increased sensitivity to Cd. They support a causative role of Cd in Itai-Itai disease among postmenopausal women in Japan. Determining the mechanism by which Cd enhances bone loss in ovariectomized animals may provide insight into mechanisms of increased bone loss after menopause in humans.